1.1 Field of the Invention
The present invention relates generally to the field of molecular biology. More specifically, it concerns novel amino acid sequences comprising the human methylthioadenosine phosphorylase (MTAP) enzyme, and the nucleic acid segments comprising the MTAP gene. Disclosed are methods and compositions related to cancer therapy involving the tumor suppressor region of human chromosome 9p21.
1.2 Description of the Related Art
1.2.1 Tumors from 9p Abnormalities
Unbalanced translocations or interstitial deletions of 9p are recurring abnormalities in a variety of tumor types including acute lymphoblastic leukemia, glioma, melanoma, non-small cell lung cancer, head and neck cancer, bladder cancer and mesothelioma (Mitelman, 1994). Homozygous deletions of DNA sequences on 9p or loss of heterozygosity have now been described in multiple tumor types (Díaz et al., 1988; 1990; Olopade et al., 1992; 1993; Kamb et al., 1994; Nobori et al., 1994). A number of the cell lines and patient samples with 9p gene deletions also lack MTAP enzyme activity. The gene encoding MTAP had not been cloned but was previously mapped to 9p22-9q13 (Carerra et al., 1984). In a few cases, the deletions that included both the IFN gene cluster and the MTAP gene were interstitial and submicroscopic, suggesting that these genes or a tumor suppressor gene closely linked to them were the target of the 9p deletions. This hypothesis was further supported by the linkage of a gene that confers susceptibility to melanoma (MLM) to 9p21 in the region between D9S126 and the IFNA gene cluster (Cannon-Albright et al., 1992).
CDKN2 (p16INK4A) was recently proposed as a candidate TSG in this locus because the gene has been shown to be rearranged, deleted or mutated in a majority of tumor cell lines (Kamb et al., 1994; Nobori et al., 1994). This gene codes for a 16 kDa protein (p16) that inhibits CDK4 and CDK6 by binding in competition with cyclin D (Serrano et al., 1993). In humans, CDKN2 is adjacent to a gene encoding a similar protein, now called CDKN2B (p16INK4B) which shares 44% homology with CDKN2 in the first 50 amino acids and up to 97 percent homology in the remainder of the protein (Hannon and Beach, 1994). Whether CDKN2 is MLM remains unclear, because two recent studies provide conflicting evidence. Hussussian et al. described six different disease-related germline mutations in CDKN2 in 33 of 36 melanoma cases from 9 families and suggested that CDKN2 likely is MLM (Hussussian et al., 1994). This is in contrast to 2/13 mutations in 9p21-linked families and 0/38 familial melanoma cases described (Kamb et al., 1994). These reports raise the possibility that CDKN2 may not be the only clinically relevant TSG on 9p and that loss of tumor suppression may involve inactivation of other as yet unidentified genes in the region in certain tumor types. Two additional regions of non overlapping homozygous deletions on 9p21 were found in malignant mesothelioma; one telomeric to CDKN2 and the other centromeric to it (Cheng et al., 1994).
The coincident loss of MTAP enzyme activity in many tumor cell lines with homozygous IFN gene deletions suggests that MTAP is closely linked to the IFN gene cluster. It was suggested that 9p TSG should be localized between the IFN gene cluster and the MTAP locus based on IFN gene rearrangements seen in two cell lines and leukemia cells from one patient with deletions on 9p (Olopade et al., 1992). In the reports published to date, it has been difficult to determine the exact position of the MTAP gene in relation to the homozygous deletions on 9p.
1.2.2 Transformation of Tumor Cells
The malignant transformation of tumor cells is known to be driven by the accumulation of various genetic alterations including numerical and structural chromosomal alterations. Among the specific alterations associated with neoplasms, the loss of tumor suppressor genes has been recognized as an important component. However, at present, deletions or mutations of the two commonly inactivated tumor suppressor genes, TP53 and RB1, have been detected only in the minority of patients with acute lymphoblastic leukemia (ALL) (Ahuja et al., 1991; Fenaux et al., 1992).
In contrast, cytogenetic deletions of chromosomal band 9p21 have been detected in 10–15% of ALL cases indicating the presence of a novel tumor suppressor gene in this region (Kowalczyk, 1983; Chilcote, 1985; Pollak, 1987). The IFN gene cluster which is located on 9p21 was found to be deleted in 43% of leukemia derived cell lines and 29% of primary leukemia samples (Díaz et al., 1988; 1990). Recently, the CDKN2 gene (p16INK4A, MTS I, CDK4I) which encodes an inhibitor of the cyclin-dependent kinase 4, has been found to be homozygously deleted in tumor cell lines and has been proposed as a candidate tumor suppressor gene in this region (Serrano et al., 1993; Kamb et al., 1994; Nobori et al., 1994). However, the frequency of point mutations of this gene in hematological malignancies has been very low suggesting that CDKN2 may be not the critically relevant gene on 9p (Quesnel et al., 1994). Moreover, the extent of the homozygous deletions on 9p have not been clearly delineated in primary tumors. 20 hematological malignancies were examined with cytogenetically well characterized 9p rearrangements to define the critical region of 9p.
1.2.3 Deletions in 9p21
There are now numerous reports of large homozygous or hemizygous deletions involving 9p21 in diverse types of tumors. The shortest region of overlap in the different tumor types varies and covers a large region on 9p21 usually from D9S171 to D9S162. Cannon-Albright et al. have recently refined their location for the melanoma susceptibility gene (MLM) to a region between D9S736 and D9S171, a genetic distance of at least 2 cM. Ishiiki et al. have also defined an interstitial deletion between D9S162 and D9S169, a region of 21 cM in the tumor from a patient with familial melanoma This region encompassed both the region homozygously deleted in melanoma cell lines and the MLM locus, and also overlapped the hemizygous germline deletion seen in lymphocytes from the patient described by Petty et al. (1993) with eight primary melanomas. In bladder cancer, a putative tumor suppressor gene locus involved in bladder tumorigenesis was localized to a 10 cM region flanked by D9S162 and D9S171 (Cairns et al., 1994). Some investigators contend that the critical region on 9p is proximal to D9S171 while others believe the region to be telomeric to the IFN gene cluster.
The high frequency of homozygous or hemizygous deletions of this region of 9p in human tumors suggests an advantage to inactivate this locus in malignant cells. Each of the three genes identified thus far in this region appears to have some biological role in cancer. Therefore, their inclusion in the deleted region may be advantageous to the malignant cell. Alternatively, intrinsic fragility or recombinogenicity around this tumor suppressor locus may make the region a hot spot for illegitimate recombination during cell division. A fragile site has been mapped to this region of 9p. Under certain culture conditions, breaks and gaps occur at a high frequency at fragile sites because they are highly recombinogenic. Although the biological consequences of fragile site expression are not fully understood, several lines of evidence point to their involvement in carcinogenesis. Therefore, it is possible that these deletions are related to the previously mapped fragile site at 9p21.
It has been suggested that the preferred mechanism for gene inactivation in this locus is homozygous deletion rather than point mutations. That there may be more than one tumor suppressor gene in this region accounting for this phenomenon is possible. If the inactivation of more than one tumor suppressor gene is required to give the cells a growth advantage, then deletions will be favored over point mutations. If two tumor suppressor genes are closely linked in a particular chromosomal region, a deletion will frequently inactivate both at the same time, while point mutations can only inactivate one at a time, and will require two of these mutations to inactivate both genes. If the genes have a low mutation rate e.g., 10−8, then the likelihood of mutating both genes is 10−8×10−8=10−16 which will be a rare event.
1.3 Deficiencies in the Prior Art
The problem of searching for additional genes on 9p21 in the face of as strong a candidate as CDKN2 seems daunting. However, the fact remains that there are still several unanswered questions regarding its role in tumor suppression. The mechanisms and genes involved in 9p deletions are complex and may not conform to the usual way of analyzing this problem. The characterization of tumor suppressor loci on 9p with regard to the genes included in the deletions would represent a major advancement in this area of tumor biology and cancer therapeutics. Determination of the mechanism(s) responsible for the propensity of this genomic region to undergo frequent deletions is critical in the development of tumor suppression therapies involving the 9p region. Likewise, DNA sequence analysis and elucidation of the amino acid sequence of MTAP would represent a major breakthrough in the development of novel gene therapies and medical diagnostics in the area of tumor suppression and cell proliferation disorder treatments.